Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. Synapses are made up of crucial cellular machineries that orchestrate a balance of membrane traffic to and from the plasma membrane. Our goal is to develop a detailed quantitative understanding of the synapse both in terms of physiological responses to action potential stimuli as well as the molecular underpinnings of its function. We recently developed sensitive approaches that allow us to characterize the heterogeneity of presynaptic function across many nerve terminals from the same cell. These new methods allow us for the first time to determine release probabilities and measures of readily-releasable pools at the single synapse level. The goal of this project is to test hypotheses about the origin of the heterogeneity of these properties. We will test the idea that this heterogeneity arises from synapse to synapse variability in two different molecular control points of neurotransmitter release. In doing so we will also obtain new and rich information about these control points at the single synapse level. The first aim will examine if the abundance of a Munc-13-1, a critical regulator of exocytosis, accounts for the heterogeneity. To do this we will examine the biophysical parameters of release at synapses in neurons derived from Munc-13-1-ECFP knockin mice. Calibration procedures will allow us to determine the absolute number of these regulatory molecules at each nerve terminal which will then be compared to functional readouts at the same terminal. We will additionally use shRNA-based manipulation of this protein will allow us to determine the molecular dose-response relationship for function at a very detailed level. Our second Aim will use this same mapping approach of presynaptic properties and determine how function is correlated with the specific types of calcium channels present at each synapse and how the abundance of functional channels influences key parameters of neurotransmitter release at the single synapse level. Finally we will examine how G-protein based modulation of synapses varies from across a population of synapses and how it impacts function. PUBLIC HEALTH RELEVANCE: Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. The functioning of the human brain relies on the careful orchestration of delivering neurotransmitter-laden vesicles to sites at nerve terminals where they can be used to deliver this chemical message on demand. Many known genetic mutations in diseases such as Parkinson's disease, migraine headache and schizophrenia are linked to proteins that control synapse function. Our work is aimed at understanding the machinery at a molecular level to better ensure the success of future therapies for these types of neuronal diseases.